Control of Cellular Processes by Transcription Factors. Studies from a diversity of prokaryotic and eukaryotic organisms suggest a gradual evolution of biochemical and physiological mechanisms and metabolic pathways. Despite different evolutionary pressures, proteins that regulate the cell cycle in yeast, plant, nematode, fly, rat, and man have common chemical or structural features and modulate the same general cellular activity. A comparison of gene sequences with known structure and/or function from one plant species, for example, Arabidopsis thaliana, with those from other plants, allows researchers to develop models for manipulating a plant's traits and developing varieties with valuable properties.
A plant's traits may be controlled through a number of cellular processes. One important way to manipulate that control is through transcription factors—proteins that influence the expression of a particular gene or sets of genes. Because transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can change entire biological pathways in an organism. Strategies for manipulating a plant's biochemical, developmental, or phenotypic characteristics by altering a transcription factor expression can result in plants and crops with new and/or improved commercially valuable properties, including traits that improve yield or survival and yield during periods of abiotic stress, improve shade tolerance, or alter a plant's sensing of its carbon/nitrogen balance.
Problems associated with water deprivation. In the natural environment, plants often grow under unfavorable conditions, such as drought (low water availability), high salinity, chilling, freezing, or high temperature. Any of these abiotic stresses can delay growth and development, reduce productivity, and in extreme cases, cause the plant to die. Of these stresses, low water availability, which in a severe form is referred to as a drought, is a major factor in crop yield reduction worldwide. Problems for plants caused by low water availability include mechanical stresses caused by the withdrawal of cellular water. Drought also causes plants to become more susceptible to various diseases (Simpson, ed. (1981) “The Value of Physiological Knowledge of Water Stress in Plants”, in Water Stress on Plants, Praeger, N.Y., pp. 235-265).
In addition to the many land regions of the world that are too arid for most if not all crop plants, overuse and over-utilization of available water is resulting in an increasing loss of agriculturally-usable land, a process which, in the extreme, results in desertification. The problem is further compounded by increasing salt accumulation in soils, which adds to the loss of available water in soils.
Water deficit is a common component of many plant stresses. Water deficit occurs in plant cells when the whole plant transpiration rate exceeds the water uptake. In addition to drought, other stresses, such as salinity and low temperature, produce cellular dehydration (McCue and Hanson (1990) Trends Biotechnol. 8: 358-362).
Salt and drought stress signal transduction include ionic and osmotic homeostasis signaling pathways. The ionic aspect of salt stress is signaled via the SOS pathway where a calcium-responsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as SOS1. The pathway regulating ion homeostasis in response to salt stress has been described-recently by Xiong and Zhu (2002) Plant Cell Environ. 25: 131-139 and Ohta et al. (2003) Proc Natl Acad Sci USA 100: 11771-11776.
The osmotic component of salt stress involves complex plant reactions that overlap with drought and/or low temperature stress responses.
Common aspects of drought, cold and salt stress response have been reviewed recently by Xiong and Zhu (2002) supra. Those include:                (a) transient changes in the cytoplasmic calcium levels very early in the signaling event (Knight, (2000) Int. Rev. Cytol. 195: 269-324; Sanders et al. (1999) Plant Cell 11: 691-706);        (b) signal transduction via mitogen-activated and/or calcium dependent protein kinases (CDPKs; Xiong and Zhu (2002) supra) and protein phosphatases (Merlot et al. (2001) Plant J. 25: 295-303; Tahtihaiju and Palva (2001) Plant J. 26: 461-470);        (c) increases in abscisic acid (ABA) levels in response to stress triggering a subset of responses (Xiong and Zhu (2002) supra, and references therein);        (d) inositol phosphates as signal molecules (at least for a subset of the stress responsive transcriptional changes (Xiong et al. (2001) Genes Dev. 15: 1971-1984);        (e) activation of phospholipases which in turn generate a diverse array of second messenger molecules, some of which might regulate the activity of stress responsive kinases (phospholipase D functions in an ABA independent pathway, Frank et al. (2000) Plant Cell 12: 111-124);        (f) induction of late embryogenesis abundant (LEA) type genes including the CRT/DRE responsive COR/RD genes (Xiong and Zhu (2002) supra);        (g) increased levels of antioxidants and compatible osmolytes such as proline and soluble sugars (Hasegawa et al. (2000) Annu. Rev. Plant Mol. Plant Physiol. 51: 463-499); and        (h) accumulation of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals (Hasegawa et al. (2000) supra).        
Abscisic acid biosynthesis is regulated by osmotic stress at multiple points. Both ABA-dependent and -independent osmotic stress signaling first modify constitutively expressed transcription factors, leading to the expression of early response transcriptional activators, which then activate downstream transcriptional activators and stress tolerance effector genes.
Based on the commonality of many aspects of low-temperature, drought and salt stress responses, it can be concluded that genes that increase tolerance to low temperature or salt stress can also improve drought stress protection. In fact, this has already been demonstrated for transcription factors, as in the case of AtCBF/DREB1, and for other genes such as OsCDPK7 (Saijo et al. (2000) Plant J. 23: 319-327) or AVP1 (a vacuolar pyrophosphatase-proton-pump, Gaxiola et al. (2001) Proc. Natl. Acad. Sci. USA 98: 11444-11449).
Advantages of a shade tolerant phenotype in commercially significant plants. In nature, plants compete for light because they grow close together. Obviously, it is disadvantageous for a plant to be positioned in the lower part of the canopy where light availability is severely limited. In response to the proximity of neighboring vegetation, many plant species have evolved mechanisms to alter dramatically their architecture in order to avoid shading by competitors. Responses to shade are many and varied, but typically shade avoidance responses are characterized by the redirection of plant resources from leaves and storage organs into increased extension growth and decreased branching. Nevertheless, there is a penalty to this mode of growth in that it often results in accelerated flowering and a reduction in the resources available for reproductive development. This is often associated with lowered seed set, truncated fruit development, and a reduction in seed germination efficiency (Giorgio-Morelli and Ruberti (2002) Trends Plant Sci. 7: 399-404). Indeed, plants often initiate shade avoidance at very early stages of development, well before restricted light availability becomes a growth-limiting factor. This can be a particular problem in row crops; at very early phases of competition when actual limitation of light is not yet an issue, plants are capable of detecting changes in the quality of horizontally reflected light from neighbors and inducing a classical shade avoidance response. In some species, lines that show a shade avoidance phenotype to far red light reflected from neighbors can show very significant yield decreases.
For crop species, planting or population density varies from crop to crop, from one growing region to another, and from year to year. Using corn as an example, the average prevailing density in 2000 was in the range of 20,000-25,000 plants per acre in Missouri, USA. A desirable higher population density would be at least 22,000 plants per acre, and a more desirable higher population density would be at least 28,000 plants per acre, more preferably at least 34,000 plants per acre, and most preferably at least 40,000 plants per acre.
The average prevailing densities per acre of a few other examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre (Cheikh, et al., (2003) U.S. Patent Application No. .20030101479). A desirable higher population density for each of these examples, as well as other valuable species of plants, would be at least 10% higher than the average prevailing density.
Problems associated with low nitrogen conditions. Nitrogen is a critical limiting nutrient for plants. However, these yield benefits have monetary and environmental costs. Nitrogen fertilizer represents a significant fraction of a farmer's input costs. For example, total fertilizer costs for corn averaged $45/acre ($111/hectare) from 1975-1995 in the U.S., (USDA-ERS (1994). in “Fertilizer Use and Price Statistics”, at http://usda.mannlib.cornell.edu/data-sets/inputs/86012/) and most of this cost represents nitrogen. Furthermore, increased fertilizer application rates are subject to diminishing returns. For instance, it was calculated that a hectare of corn retains 39% of the first 100 kilograms of nitrogen applied as fertilizer, but only 13% of the second kg of nitrogen applied (Socolow (1999) Proc. Natl. Acad. Sci. USA 96: 6001-6008). Nitrogen fertilizer that is not taken up by plants is generally lost as runoff or converted to nitrogen gases by microbial action, contributing to water and air pollution. Improving the nitrogen use efficiency of crop plants has the potential to reduce fertilizer application rates, providing both cost savings and environmental benefits.
Nitrogen uptake and assimilation are regulated at many stages by the total carbon and nitrogen status of the plant (reviewed in Glass et al (2002) J. Exp. Bot. 53: 855-864; Coruzzi et al. (2001) Curr. Opin. Plant Biol. 4: 247-253; and Stitt et al. (2002) J. Exp. Bot. 53 (370): 959-970). Transcriptional, translational, and post-translational regulation have been demonstrated. These regulatory effects are mediated through sensing of nitrate, amino acids, and sugars.
Nitrate and ammonium transporters are transcriptionally up-regulated in response to external concentrations of substrate and down-regulated in response to glutamine, and are also thought to be post-translationally regulated by cytosolic nitrate and ammonium concentrations (Glass et al (2002) supra; Coruzzi et al. (2001) supra), Nitrate reductase in tobacco is transcriptionally regulated by sugars and nitrate, translationally regulated by light (or products of photosynthesis), and post-translationally stabilized and activated in the light (Stitt et al. (2002) supra).
Glutamine synthetase is transcriptionally induced by sucrose and light, and repressed by glutamate, glutamine, aspartate, and asparagine (Oliveira et al. (2002) Plant Physiol. 129: 1170-1180). Conversely, asparagine synthetase is generally repressed by light and sugars. GOGAT transcription and activity are also thought to be regulated by nitrate and sugars, respectively (Stitt et al. (2002) supra). The stability of a number of proteins involved in nitrate assimilation, including nitrate reductase and cytosolic glutamine synthetase, was found to be regulated by sugar through the action of 14-3-3 proteins (Cotelle et al. (2000) EMBO J. 19: 2869-2876; Finnemann et al. (2000). Plant J. 24: 171-181).
Plant nitrogen use efficiency may be increased by several possible mechanisms (Lawlor (2002) J. Exp. Bot. 53: 773-787), including increased nitrogen uptake through higher root surface area, deeper penetration into the soil, or more high affinity nitrate or ammonium transporters, increased assimilation, possibly by increasing activity of assimilatory enzymes or removal of negative regulation, or increased capacity to store nitrogen when it is available. Nitrogen is stored in the form of nitrate in cell vacuoles, but stored nitrate supplies are exhausted in a matter of days (Glass et al. (2002) supra). Nitrogen is also stored in the form of amino acids and protein, and this storage is dependent upon sufficient carbon availability. Control of nitrogen losses is also possible. Nitrate and ammonia exit as well as enter root cells. Photorespiration is another source of ammonia loss. Ammonia released through photorespiration is recycled through the GS/GOGAT pathway, but this process may not be fully efficient. Overexpression of cytosolic glutamine synthetase in tobacco increases biomass, presumably through increased efficiency of ammonia recycling (Oliveira et al. (2002) supra). Finally, the intrinsic nitrogen use efficiency (defined as biomass produced per unit N) could be changed by changing the plant's fundamental carbon/nitrogen ratio.
Using anthocyanin accumulation as an indicator of nitrogen limitation stress Lam et al. (Lam et al. (1994) Plant Physiol. 106: 1347-1357) showed that the young seedlings of 35S-ASN1 lines are less stressed under nitrogen-limiting conditions, accumulating less anthocyanin compared to wild-type seedlings grown under the same conditions. In the 35S::ASN1 lines, an increase of ASN1 mRNA was accompanied by increases in free asparagine, soluble seed protein and total seed protein compared with control seedlings. It Previous attempts to manipulate seed storage protein genes have resulted in altered protein composition but not in overall nitrogen storage in seed (babe et al. (2002) Curr. Opin. Plant Biol. 5:212-217).
We have identified numerous polynucleotides encoding transcription factors, functionally related sequences listed in the Sequence Listing, and structurally and functionally similar sequences, developed numerous transgenic plants using these polynucleotides, and analyzed the plants for their tolerance to shade, drought stress, and altered carbon-nitrogen balance (C/N) sensing. In so doing, we have identified important polynucleotide and polypeptide sequences for producing commercially valuable plants and crops as well as the methods for making them and using them. The present invention thus relates to methods and compositions for producing transgenic plants with improved tolerance to drought and other abiotic stresses, with altered C/N sensing, and/or with improved tolerance to shade. This provides significant value in that the plants may thrive in hostile environments where low nutrient, light, or water availability limits or prevents growth of non-transgenic plants. Other aspects and embodiments of the invention are described below and can be derived from the teachings of this disclosure as a whole.